Hi everyone. I have experience using the CRISPR-Cas9 system to establish knockout cell lines. Now, I am planning to introduce a single point mutation into my gene of interest using the CRISPR system, and I really need some help. The main problem is that I don't know how to design and construct related plasmids. Many thanks for any relevant assistance.
Instead, I have experience with lentiviral packaging, cell screening, and the identification of monoclonal. If needed, I may be able to assist you in these areas. Thank you kindly for your help!
You are probably better off using a Cas9 derived base modifier (Reviewed in Article Crispr-cas9 dna base-editing and prime-editing
).
Dear Robert Adolf Brinzer
Thank you so much for your suggestion, and this article is very useful.
Wangsheng
Hi,
it may be not possible for you to introduce your desired change using Base Editing, as sugessted above (depending on the change). If so, please have look on Horizon products https://horizondiscovery.com/-/media/Files/Horizon/resources/Selection-guides/edit-r-gene-editing-workflow-guide.pdf
We used the following approach: Cas9 plasmid, gene-specific crRNA, common tracrRNA and gene-specific ssDNA (single-stranded DNA) containing the mutation of interest (so basically ssDNA works as a template to repair after Cas9 cut). You have to design your own crRNA and ssDNA specific for the gene; Horizon website has an oligonucleotide creator which helps. You can expect ~10% of efficiency in case of knock-in generation, but it can be cell line-dependent.
Good luck
Hi Malgorzata Grudzien
Thank you very much for your help. I will try to use the method you provided.
Wangsheng
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Molecular Methods
- Cloning